Nicotiana benthamiana, with BaMV or superinfects a transgenic N. benthamiana expressing the full-length BaMV genome. Single-nucleotide polymorphisms (SNPs) of satBaMV populations were analyzed by deep sequencing. Forty-eight SNPs were identified across four different experimental treatments. Most SNPs are treatment-specific, and some are also ephemeral. However, mutations at positions 30, 34, 63, and 82, all located at the 5′ untranslated region (UTR), are universal in all treatments. These universal SNPs are configured into several haplotypes and follow different population dynamics. We constructed isogenic satBaMV strains only differing at positions 30 and 82 and conducted competition experiments in protoplasts and host plants. We found that the haplotype that reached high frequency in protoplasts and inoculation leaves also exhibited poor dissemination to systemic leaves and vice versa, thus suggesting an apparent trade-off between local replication and long-distance dissemination. We posit that the trade-off is likely caused by antagonistic pleiotropy at the 5′ UTR. Our findings revealed a hitherto under-explored connection between satRNA genome replication and movement within a host plant. The significance of such a connection during satRNA evolution warrants a more thorough investigation.Satellite RNAs (satRNAs) are molecular parasites that depend on their non-homologous helper viruses (HVs) for essential biological functions. While there are multiple molecular and phylogenetic studies on satRNAs, there is no experimental evolution study on how satRNAs may evolve in common infection conditions. In this study, we serially passaged the Bamboo mosaic virus (BaMV) associated-satRNA (satBaMV) under conditions in which satBaMV either coinfects an uninfected host plant,  Satellite RNAs (satRNAs) are a group of subviral agents that were first discovered by the presence of an extra, slower-sedimenting band during the ultracentrifugation step for the purification of the tobacco ringspot virus (TRSV) . Howeverde novo evolution of satRNA sequences  was initially isolated from a BaMV-infected bamboo  gene. A homozygous second filial generation (F2) of the symptomatic line 27–17 was used in this study  of four inoculated plants were collected and pooled together at 10 days post-infection (DPI) and designated as IL passage 1 (IL-P1). The systemic leaves  were collected and pooled at 30 DPI and designated SL-P1. The pooled leaves were cut into small pieces for further processing. Half of each P1 sample was frozen with liquid nitrogen and stored at −80°C for satBaMV population analysis. The other half was immediately ground with 10 volumes of sodium phosphate buffer  with a pre-chilled mortar and pestle. The resulting crude sap was then used to inoculate four N. benthamiana plants for the next round of infection with mechanical means. Leaf samples were collected at 10 DPI for IL and 30 DPI for SL, as described above, and designated P2. A total of 10 passage (P1–P10) samples were collected.Experimental designs for the serial passage experiments are shown in  or pKF4  on four Total RNAs were extracted from 0.1 g frozen leaf samples using TriPure Isolation Reagent, following the manufacturer’s instructions . RNA quality and quantity were determined by NanoDrop ND-1000 Spectrophotometer . RNA blot was performed following the instruction of DIG DNA Labeling and Detection Kit . Briefly, 1 μg of total RNA was separated on a 1% SeaKem® LE agarose gel  in 0.5X TBE buffer . RNA was then transferred onto a Hybond-N+ nylon membrane . Transferred blots were hybridized with DIG-labelled probes against BaMV CP-3′ UTR or BSF4 genomes . BaMV orin vitro transcripts. The resulting first-strand cDNAs were then used as templates for generating full-length satBaMV amplicons by PCR using primers A02-R and A01-F (targeting the 5′ end of the satBaMV genome)  and primer A02-R (targeting the 3′ end of the satBaMV genome). For the control sample (Tx), total RNA from healthy plants was spiked with 100 ng BSF4  genome) . PCR reaWhile all relevant samples in each passage were collected, limited samples were subjected to deep sequencing due to limited resources. The analyzed samples are (1) P1, P9, and P10 for the Co-SL passage, (2) P1, P9, and P10 for the Su-SL passage, (3) P1, P2, P6, and P10 for the Co-IL passage, and (4) P1, P2, P6, and P10 for the Su-IL passage. The prefix “Co” denotes the viral populations derived from coinfection of both BaMV and satBaMV, while the “Su” populations were obtained from superinfection of the transgenic line 27–17 plants.The Illumina raw reads were preprocessed using Trimmomatic 0.35  to removPlasmid pCF4 is an infectious cDNA clone of satBaMV BSF4, the expression of which is driven by the 35S promoter . QuickChN. benthamiana leaves  and its associated satellite RNA (satBaMV) as the model system and V genome . As showV genome , lane 6.To realize the coinfection scenario, we used the agroinfiltration method to introduce plasmids expressing satBaMV and BaMV into uninfected wild-type host plants. To simulate the superinfection scenario, the plasmid expressing satBaMV was agroinfiltrated to the transgenic line 27–17. As shown in Prompted by the observation that satBaMV replicates differentially under coinfection and superinfection conditions, we decided to investigate how satBaMV would evolve under these two infection scenarios we envisioned. Serial passage experiment (SPE), a common practice for studying evolution in a laboratory setting, was adopted for this purpose. Besides evolution in different infection conditions, we also expanded our study to include the contrast between evolution under the condition of local replication vs. long-distance dissemination. The rationale is that during either vector or mechanical transmission of plant viruses, the source of the transmitted viruses is often from systemic leaves that are some distance away from the initial inoculation site to which the original viral population is introduced.P20 gene when comparing local replication and long-distance dissemination scenarios.We hypothesized that the selective pressure on satBaMV could be quite different between Co and Su or between local replication (using “IL” as a proxy) and long-distance dissemination (using “SL” as a proxy). Since the accumulation of the satBaMV is reduced more under the Su than the Co condition , this obLeaf samples were collected during the passage experiments. The satBaMV populations were extracted, reverse-transcribed, amplified with PCR, then deep-sequenced.Analyses of the deep-sequencing results showed that more than 99% of the reads are unambiguous and can be mapped to the BSF4 genome, with an average read-depth of 376,251 per site  .To establish the baseline of sequencing noise resulting from nucleotide misincorporations during reverse transcription, PCR amplification, and deep sequencing, we included a spiked control sample with RNA transcripts derived from a cDNA clone of the ancestral satBaMV strain, BSF4 (GenBank accession number: AY205227). For example, in our control sample, the read-depth at position 82 is 515,395; 174 showed the U82A, 1,199 the U82C, and 851 the U82G reads, with frequencies of 0.0003, 0.0023, and 0.0017, respectively. The combined misincorporation rate at this particular position is therefore summed up to be 0.0043. On average, for the satBaMV genome, the combined misincorporation rate per nucleotide position is 0.0026 ± 0.0017 (std. dev.) with a range of 0.0004–0.0214. Interestingly, the observed misincorporation rates at each nucleotide position vary greatly along the entire satBaMV genome, with the elevated rates clustered at the 5′ and 3′ UTRs see .To identify sequence variations that are likely due to evolutionary processes rather than nucleotide misincorporations during sample preparations, we applied an arbitrary but conservative criterion by defining a specific base-calling a single-nucleotide polymorphism (SNP) if the base-calling rate of any particular variant—after discounting the corresponding rate in the control sample—is ≥0.01. For example, for the Co-IL-P1 sample, the uncorrected frequency for U82C mutation is 0.0677, while the corresponding change in the control sample is 0.0023. The corrected frequency for the U82C mutation is then calculated as 0.0654 for this specific sample (satBaMV population). The exceptions are positions 697, 749, 768, 807, 817, and 828, all of which have misincorporation rates ≥0.01 and thus are excluded from the analyses.Based on the criterion established above, we identified 48 SNPs among 45 sites that have net frequencies  that are ≥0.01 see .A closer inspection of the SNP distribution among the experimental sets of Co-IL, Co-SL, and Su-IL/Su-SL see  revealedP20 region, 28 only appeared in one of the satBaMV populations; none appeared in all. Furthermore, many SNPs are ephemeral and appear only once in the sampled time points, often in the intermediate passages and at relatively low frequencies. The exceptions are U215G (resulting in P20 I19S change) and U317C (resulting in P20 V53A change) in the Co-IL populations. These two SNPs showed increased frequencies from ∼1% in P6 to ∼20% in P10. Another exception is the U661C mutation (resulting in P20 Y168H change) of the Su-IL/Su-SL set. This SNP is maintained from P6 to P10 at ∼75–85%. Most SNPs in the 3′ UTR are ephemeral and relatively low in frequencies. The only exception is the G799A mutation in the Su-IL/Su-SL set, with its frequency increased from ∼5% to ∼25% within one passage.Of the 36 SNPs in the P20 gene, that are only found in the Su sample but not the Co samples, and (2) there is one SNP (U30A) that is only found in the IL samples, but not the SL samples.The distribution of all 48 SNPs among the SPEs is summarized as a Venn diagram, shown in Since positions 30, 34, 63, and 82 in the 5′ UTR are close to each other and approximately a quarter  of mapped sequencing reads covered all four sites, we were able to obtain haplotype frequencies in this specific region among all passage experiments without ambiguity. Only haplotypes that have once reached ≥0.01 in frequency are recorded (after correction for nucleotide misincorporation as described previously). The haplotype frequencies are shown in de novo in SL.Of 256 possible permutations, 16 haplotypes  were identified among all SPEs passages . Each CoTemporally, we can discern three groups of haplotypes in all SPEs . The earThe intermediate haplotype group showed a rise-and-fall pattern during the passages , then we should expect the rankings of genotype frequencies to be concordant between the IL and SL sampling sites.For the Co condition, the dominant haplotypes (with frequencies ≥0.1) in P10 are U34C/U63G (0.377), U63G (0.294), and U30A/U82A (0.217) for the Co-IL passage and U30C (0.640) and U30C/U82C (0.326) for the Co-SL passage . These tFor the Su condition, however, the result is different. While samples from the Co-IL and Co-SL passages are independent, the samples from the Su-IL and Su-SL are temporally linked see . The Su-So far, comparisons of satBaMV haplotypes in the 5′ UTR revealed a possibility that mutations in this region could be beneficial to persistence in a local area but detrimental to long-distance dissemination. That is, genetic constraint, in the form of antagonistic pleiotropy, in the 5′ UTR  could generate a trade-off between local replication and long-distance dissemination.To determine whether mutations we observed in the 5′ UTR during SPEs  exhibit N. benthamiana protoplasts supplied with pBaORF1 (providing BaMV’s RdRp) were separately infected, we found that each haplotype can accumulate to similar levels of genome RNA (We used the UNAFold servicenome RNA  and P20 nome RNA  as thoseN. benthamiana protoplasts (in planta inoculation leaves (IL) and systemic leaves (SL).Together with the ancestral BSF4 (UU), these five satBaMV strains were tested for their replication competence, local replication, and long-distance dissemination in (1) toplasts  and (2) N. benthamiana protoplasts. The competition experiment was conducted by transfecting replicase-expressing protoplasts (the same as the single infection experiments) and mixtures with an equal amount of plasmids that encode each of the five satBaMV strains (providing satBaMV RNA transcripts). To determine the competition outcome, we sampled the resulting satBaMV population by cloning and determined the identity of each individual via sequencing. The competition experiment was repeated three times. Although only a small number of clones (18–24) were sequenced, Fischer’s exact test of independence showed that the ratios of these haplotypes were not significantly different among the three replicates (p = 0.5437), thus allowing the pooling of the data for a better statistical analysis. An exact test of the goodness-of-fit of the pooled data showed that the ratio of haplotype frequency significantly deviates from the expected equal ratio , indicating differential replication competence among these haplotypes during mixed infection. Post hoc analysis using the exact binomial test showed that both the ancestral BSF4 [0.015 ] and U30A/U82A [0.076 (0.025–0.168)] have lower than expected frequencies after the 48-h incubation period while the haplotype U82C has a higher-than-expected frequency [0.318 (0.209–0.444)]. The other two haplotypes, U30C/U82C [0.303 (0.196–0.429)] and U30A/U82C [0.288 (0.183–0.413)] showed frequencies as expected . (1) Competition in in planta. To test whether these haplotypes differ in their ability to persist in the IL and long-distance dissemination to SL, we co-inoculated BaMV and an equal proportion of satBaMV mix onto N. benthamiana using agroinfiltration. Plant tissues were collected at the same intervals as those used during the SPEs, i.e., 10 DPI for IL and 30 DPI for SL. The frequency of each haplotype is again sampled with cloning and enumerated by sequencing. After Fisher’s exact test of independence, showing non-significance , the number of each haplotype from all three replicates was pooled together. An exact test of goodness-of-fit showed that both haplotype ratios significantly deviate from the expected equal proportion . The exact binomial test was used to conduct post hoc analyses and determine the estimated proportion for each haplotype.(2) Competition Among these haplotypes, the ancestral BSF4 showed a consistent decline in its frequency in both the IL and the SL . Two haplotypes showed opposite frequency trends in the IL and SL conditions: U82C has a higher frequency in IL [0.343 (0.271–0.422)] but lower in SL [0.037 (0.014–0.078)], while U30A/U82A has the opposite trend . The rest two haplotypes showed a similar pattern of remaining as expected in one condition while displaying an increased or decreased frequency in the other. For U30C/U82C, its frequency remained as expected in the IL condition [0.147 (0.097–0.211)] but declined in the SL condition [0.110 (0.067–0.169)]. For U30A/U82C, its frequency increased in the IL condition [0.417 (0.341–0.497)] but remained the same in the SL condition [0.245 (0.182–0.319)]. These competition experiments showed that the ancestral BSF4 is at a competitive disadvantage in the presence of other mutations in positions 30 and 82. Its lower replication competence in protoplasts is directly translated to a lower presence in the IL  and SL. Some mutations exhibit a trade-off of home (IL) versus away (SL) advantages. For example, U82C mutation has a higher replication competence and is likely translated into its advantage in persisting in the IL. But such an advantage becomes a disadvantage when the systemic leaves are examined. The complete opposite is true for the U30A/U82A haplotype, which has a lower replication competence and thus is translated to a disadvantage in the IL. But, somehow, such a disadvantage becomes a great advantage for long-distance dissemination to the SL. This unexpected result suggests that the advantage of long-distance dissemination is not simply achieved by having more replications in the IL, followed by dissemination to the SL. A different mechanism(s) is needed to explain how different satBaMV strains compete to be disseminated to the entire plant.in situ. Such is the caricatured “reality” a satRNA will face.To ensure an effective transmission from one plant host to another, we envision that a satRNA will need to fulfill a phenotypic demand—namely, maintaining as high a population frequency as possible throughout the entire infected plant . We reason that any genotype that can meet such a demand will have a higher chance of being transmitted to the next host plant. Consequently, the selective pressure, as a first approximation, is likely to be exerted on two aspects of a satRNA’s biology: genome replication and within-host dissemination; both activities depend on the resources  provided by its cognate HV . TherefoIt is not clear which infection scenario is more advantageous to an invading satRNA; however, our study showed that the accumulation of satBaMV is suppressed more under the superinfection condition see , suggestNevertheless, the comparison between infection scenarios yielded an unexpected observation: the frequency of ancestral BSF4 haplotype declined faster and to a lower extent under the Co scenario see . This obcis-acting element of the BaMV genome, e.g., the 5′ UTR, that allows the BaMV more efficient sequestration of the replicase, or in BaMV’s replicase gene, in which mutations may render the replicase more discriminatory against satBaMV’s 5′ UTR. Both evolutionary pathways can potentially result in a more stringent molecular environment for satBaMV replication. But surprisingly, genome-wide sequencing of the corresponding BaMV populations under the coinfection scenario revealed that the overall genomic compositions of the BaMV populations remained relatively constant. For example, for the four Co-IL populations, the proportions of the BaMV genome that are polymorphic progressed from 0.00% (P1), 0.05% (P2), 0.55% (P6), to 0.61% (P10), and for the three Co-SL populations, the proportions are 0.14% (P1), 0.75% (P9), to 0.50% (P10) (see ORF1 (6), and ORF2 (1) genes. However, closer examination of these ORF1 and ORF2 SNPs showed that none results in amino acid substitution. Therefore, the replicase protein will be identical to that of the ancestral BaMV strain. The only remaining near-fixation SNP at position 40 is located at the base of the putative ASHL, an important feature that contributes to replication competence  see . MoreoveP10) see . These nmpetence . Since oIn summary, the seemingly convergent evolution of satBaMV’s 5′ UTR under both the coinfection and superinfection scenarios and the lack of BaMV evolution under the coinfection scenario suggest that the two seemingly different selective environments  are quite similar to each other, at least from the point-of-view of satBaMV. These results further imply that the dynamics of satBaMV’s haplotypes at the 5′ UTR may be mainly driven by competition within the satBaMV population rather than competition between satBaMV and BaMV populations.Antagonistic pleiotropy is a phenomenon in which a mutation is beneficial for the carrier in one life stage (or environment) but detrimental in another ,b. The cIn our current study, comparing satBaMV populations between IL and SL revealed a striking pattern of 5′ UTR haplotypes that dominated either IL or SL. We interpreted the pattern of contrasting differences as being consistent with the hypothesis that there is a trade-off between local replication and long-distance dissemination. That is, genotypes that are better at genome replication are worse at long-distance dissemination, and vice versa. We tested this hypothesis by competing five isogenic satBaMV strains  that only differed in positions 30 and 82 see , as reveWhat mechanism(s) could have contributed to the apparent trade-off between replication and movement? It has been established that the systemic movement of the BaMV RNA genome is in the form of a ribonucleoprotein (RNP) complex that comprises at least viral-encoded MPs  and CP . We can in situ. Despite being evolutionarily transient, these movement-advantageous genotypes could nevertheless impact satBaMV evolution. During the initial stage of infection, the preferentially trafficked satBaMV genotypes are like early founders who bring with them not only the movement-advantageous mutations but also all other mutations in the genome that happened to hitchhike along. Such a genetic hitchhiking may contribute to the overall satBaMV sequence diversity even if the replication-advantageous mutations eventually outcompeted the original movement-advantageous mutations.Although we have identified the presence of movement-advantageous genotypes, we believe that these genotypes may be short-lived. That is, given a long enough time, coupled with the high mutation rate characteristic of a quasispecies population, the mutations responsible for movement advantage will quickly be replaced by mutations, such as the U82C, that confer higher replication rates. Therefore, for a chronic infection like satBaMV, mutations that allow for better movement will eventually be replaced by mutations responsible for better replication, irrespective of whether the replication-advantageous mutations are trafficked from the initial infection site or eventually emerge N. benthamiana tobacco plant, which is significantly different from the natural bamboo hosts. Nevertheless, our proposed trade-off between local replication and long-distance dissemination may still operate in the bamboo hosts, albeit with a different set of antagonistic pleiotropy mutations. It will be of great interest to determine whether satBaMV also exhibits a similar trade-off in a monocot host, such as Brachypodium distachyon  can be found below: S-CL, M-RL, Y-HH, I-NW, and N-SL contributed to conception and design of this study. S-CL and M-RL performed the experiments. S-CL and I-NW organized and analyzed the amplicon datasets and results. I-NW performed the statistical analysis. S-CL, I-NW, and N-SL wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Thematic Research Program from Academia Sinica, Taiwan (AS-103-TP-B01) to N-SL and I-NW and the Academia Sinica Investigator Award, Taiwan (AS-IA-108-05), to N-SL.The authors would like to thank Mei-Yeh Lu  for performing Illumina sequencing, Wen-Dar Lin  for bioinformatics analysis, Mei-Jane Fang  for Sanger DNA sequencing analysis, and Jhih-Wei Wang and Meng-Hsun He for their technical supports.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The incidence of high-grade anal intraepithelial lesions (HSILs) has increased in recent years among men who have sex with men with human immunodeficiency virus (HIV). This work evaluated the validity of the human papilloma virus viral load (HPV-VL) versus cytological and qualitative HPV results to detect HSILs.κ) were also calculated.From May 2017 to January 2020, 93 men who have sex with men and HIV were included in an anal cancer screening program from the Infectious Diseases Unit at a tertiary-care hospital in Alicante (Spain). The gold-standard for the screening of anal HSILs is the anal biopsy using high-resolution anoscopy. The diagnostic methods compared against gold-standard were HPV-16-VL, HPV-18-VL, and HPV-16-18-VL co-testing, anal cytology, and qualitative HPV detection. The receiver operating characteristic (ROC) curve and cut-off points for HPV-VL were calculated. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen’s Kappa coefficient (n = 18) HSIL, 29.1% (n = 27) LSIL, and 51.6% (n = 48) negative. An HPV-16-VL >6.2 copies/cell was detected in the HSIL biopsy samples (p = 0.007), showing a sensitivity of 100% and a specificity of 46.2%. HPV-18-VL and HPV16-18-VL co-testing showed a sensitivity of 75% and 76.9% and a specificity of 72.7% and 61.3%, respectively. The highest PPV was 50% obtained with the cytology and HPV-18-VL. The HPV-16-VL showed a NPV of 100%, followed by 88.9% in the HPV-18-VL and 87% in the abnormal cytology. Cohen’s Kappa coefficient were: HPV-18-VL (κ = 0.412), abnormal cytology (κ = 0.353) and HPV-16-VL (κ = 0.338).The mean patient age was 44.6 ± 9.5 years. All of them received antiretroviral treatment, 96.8% had an HIV viral load of <50 copies/mL and 17.2% had a previous diagnosis of AIDS. The diagnosis of the anal biopsies were: 19.4% (HPV-VL testing improved the detection sensitivity but not the specificity for HSIL biopsies compared to anal cytology and the qualitative detection of HPV. In men who have sex with men and HIV the HPV-VL could be an useful tool for diagnosis of HSILs in anal cancer screening programs. Further studies will be needed to evaluate the clinical implications of HPV-VL in these programs. Anal cancer is a rare digestive tumour type whose incidence has increased in recent years , especiaAnal biopsy using high-resolution anoscopy (HRA) is currently considered the gold standard for the diagnosis and management of HSIL . HoweverThe validity of these methods for the detection of anal precursor lesions or anal cancer in people with HIV and MSM was evaluated in a meta-analysis . For anaGiven the wide variability in the results published on the sensitivity and specificity of the different screening tests, new biomarkers such as HPV E6/E7 mRNA, p16, and Ki67 have been explored that could help in the screening of HSILs. The sensitivity of these tests for the detection of HSILs is also variable; for E6/E7 the sensitivity ranged from 69.6% to 71% and the specificity from 56.1% to 55.6%, while for p16 or p16/ki67, a sensitivity of 38.1% to 90% and a specificity of 50.5% to 87.9% was obtained . As we cDetermination of the HPV viral load (HPV-VL) is another biomarker directly associated with the severity of cervical intraepithelial lesions in cervical cancer screening , with thTo the best of our knowledge, HPV-VL has not been studied in the population of MSM with HIV in Spain. Therefore, considering the good predictive results of the majority of published international studies, we decided to study the ability of the HPV-VL to detect high-grade anal lesions in a population of MSM with an HIV infection who continued clinical monitoring at our clinic. The objective of this study was to evaluate the validity of using the HPV-VL to detect HSILs in men with HIV who have sex with men compared to the efficacy of qualitative and cytological HPV test results.This was a cross-sectional study of a diagnostic test evaluation.Eligible candidates for anal cancer screening were included from the Infectious Diseases Unit at a tertiary-care hospital in Alicante (Spain). Men who attended an anal cancer screening test between May 2017 and January 2020 were consecutively selected to participate in this study. All of them met the following inclusion criteria: males aged 18 years or older, a confirmed HIV infection, an HIV transmission mechanism of MSM sexual relations, and participating in the anal cancer screening program. The only exclusion criterion was having been previously vaccinated against HPV.ad hoc structured data collection questionnaire was prepared for data collection. Sociodemographic variables (sex and age), HIV-related clinical variables , undetectable HIV viral load [HIV-RNA <50 copies/mL], CD4+ nadir, current CD4+ cell value/µL, CD4/CD8 ratio and presence of genital warts), toxic habits , and variables related to sexual habits  were collected.An Anal cytology was performed on all the participants using the specific cytobrush material which was later transferred to ThinPrep sample preservation jars®. This technique involves introducing a brush into the anal canal using enveloping movements from the perianal skin up to four cm above the anal margin. Subsequently, this brush was introduced into a liquid medium for a few seconds  for preservation and analysis. Analysis of the anal cytology results was first performed by a single pathologist specialising in the diagnosis of anal dysplasia. The Bethesda classification was used  with theThe anal cytology was subsequently sent to the clinical microbiology service to determine the viral load and qualitative detection of HPV. The viral load of HPV-16 and HPV-18 was determined and the viral load co-testing of HPV-16-18 was performed taking the highest quantified value of HPV-16 or HPV-18 as the reference. Detection of human genomic DNA in the HPV viral load was performed using TaqMan™ Control Genomic DNA . The results were normalised and expressed as viral copies/human cell (copies/cell). The qualitative detection of HPV was carried out with the Cobas® 4800 HPV-Test. The results obtained were for HPV-16 and HPV-18, as well as a combined method for 12 high-risk human papillomavirus genotypes (HR-HPV), including 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.The HRA was performed on all the participants by the same team which comprised a proctologist and a nurse, both trained in performing this technique. The anoscopy was carried out in an operating room set up for this purpose. First, a digital rectal examination and visual examination were performed to detect macroscopic changes. Second, a transparent plastic anoscope (THD® N-Ano) was introduced into the anal canal for visualisation purposes. Using a colposcope with a light source and high magnification binocular vision, the anal tissue was then visualised. Afterwards, a swab wrapped in gauze and impregnated with 3% acetic acid (Lugol’s stain) was introduced for 2 min and was withdrawn; the anoscope was then reintroduced to visualise the anal tissue. After staining, areas likely to be preneoplastic—acetowhite positive—were sought. When necessary, a further 5% Lugol staining was performed with a swab to improve visualisation of the possible lesions. Third, an anal biopsy was taken from areas suspected of a lesion using Baby Tischler forceps and was then sent to the pathology department for analysis. When a patient had multiple areas biopsied on the same day, the area with the highest degree of dysplasia was used for analysis. The histological classification of the anal biopsy for its diagnosis was based on the Lower Anogenital Squamous Terminology Project , with thAll the participants were informed of the reason for the screening and signed an informed consent form for their participation in the study and for data collection. Participants were informed during HIV medical follow-up visits of the simultaneous performance of: anal cytology, qualitative HPV detection and high-resolution anuscopy with anal biopsy. These results were analysed by the Pathological Anatomy Service. The Clinical Microbiology Service subsequently analysed the viral load and carried out the qualitative detection of HPV.SD) and of the categorical variables using the absolute frequencies and percentages. Anal biopsy using HRA was used as the gold standard method of screening for HSILs. In the statistical analysis of the association and comparison of the different diagnostic tests, the histological result of HSIL versus non-HSIL was used. The latter included negative anal biopsy, LSIL, and anal warts. Thus, the continuous variables were analysed using Student t-tests or Mann–Whitney U tests, depending on the normality of the variables . Categorical variables were analysed using chi-squared tests and Fisher tests for association studies. In all the analyses, the value of p < 0.05 was considered statistically significant.A descriptive analysis of the continuous variables was performed using the mean and standard deviation (κ) were calculated to compare the usefulness of the different screening methods: HPV-16, HPV-18, HPV-16-18 co-testing, anal cytology, and the qualitative determination of HPV. Taking into account the high prevalence of anal HSILs data found in other studies  curves, together with the area under the curves (AUCs), were calculated to detect the cut-off point of the viral load of HPV-16, HPV-18, and the HPV-16-18 co-test to differentiate between the diagnosis of HSILs and non-HSILs by HRA biopsy. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen’s Kappa coefficient . All the participants were informed about the study and authorisation was requested by written informed consent before their participation. The confidentiality and anonymity of the data was ensured in accordance with current legislation on the protection of personal data and guaranteed rights (LOPD 03/2018 of December 5) and EU Regulation 2016/679 of the European Parliament.From May 2017 to January 2020, a total of 103 patients were seen at a tertiary-care hospital anal cancer screening clinic: 93 men and 10 women. A total of 93 participants who were MSM with an HIV infection were included in this work. Sociodemographic variables, clinical variables related to HIV, toxic habits, and sexual habits were collected of all the participants . The mean = 18) presented a HSIL, 29.1% (n = 27) a LSIL, and 51.6% (n = 48) a negative result. The quantification of the viral load in the HPV-16-18 co-test was higher for the total number of participants compared to HPV-16 and HPV-18 (41 copies/cells vs. 28.9 copies/cells vs. 2.16 copies/cells). In the anal cytology, 17.2% (n = 16) of the participants had a cytological alteration  while 81.7% (n = 76) obtained a negative result. There was one patient whose cytological result could not be obtained because insufficient material had been obtained. The highest prevalence of HPV detected was HR-HPV accounting for 83.7% (n = 77), followed by HPV-16 at 40.2% (n = 37), and finally HPV-18 with 17.4% (n = 16). In the comparisons made between participants with HSIL versus non-HSIL anal biopsy results, there were significant differences in HPV-16-VL  and the HPV-16-18 co-test viral load . In the rest of the variables studied, significant differences were found in abnormal anal cytology (p = 0.001), qualitative HPV-16 (p = 0.044), and qualitative HR-HPV (p = 0.037).A descriptive analysis of the different diagnostic tests studied for anal cancer screening was performed using HSIL and non-HSIL anal HRA biopsy for comparison . In the κ = 0.412) showed the highest Cohen’s Kappa coefficient followed by abnormal cytology (κ = 0.353) and HPV-16-VL (κ = 0.338). However, the poorest results were in the qualitative HR-HPV (κ = 0.037) and the qualitative HPV-18 (κ = 0.063).The results regarding the validity of HPV-VL, anal cytology, and qualitative HPV detection for the diagnosis of anal HSIL in participating HIV positive MSM were compared . The besThe aim of this study was to evaluate the validity of HPV-VL determination, compared to cytological and qualitative HPV results, for the diagnosis of HSILs using the histological results of anal biopsies as the gold standard. HPV-16-VL showed the highest sensitivity for the diagnosis of HSILs in anal cancer screening, although with a low specificity. HPV-18-VL showed the most balanced results between sensitivity and specificity with the drawback that only a few participants were studied. However, HPV-16-18-VL co-testing produced the most similar sensitivity and specificity results compared to conventional methods of anal cytology and qualitative HPV detection.As already observed in previous studies in HIV-infected men , the anaIn contrast, in our sample there was a high prevalence of qualitative HPV. The prevalence of HPV-16 was around 40%, similar to that found in other studies , which wvs. 100% vs. 76.9%, respectively) but high specificity . A meta-analysis reported a detection sensitivity of 85.2% and a specificity of 52.8% for anal cytology of HSILs , although the specificity was still improved (61.3% vs. 46.2%). It is possible that including HPV-18-VL with HPV-16-VL to obtain a combined result will improve the diagnostic validity of viral load in the diagnosis of HSILs. In addition, HPV-18-VL produced results with a lower discrepancy between sensitivity and specificity, at 75% and 72.7%, respectively. However, these data must be interpreted with caution because very few cases with HPV-18-VL and HSILs have been studied, and so these results are difficult to extrapolate. Notwithstanding, other studies have suggested that HPV-18 could also be a useful biomarker for the detection of anal lesions (Nonetheless, the sensitivity of the HPV-16-18-VL co-test was lower than that of the HPV-16-VL (76.9%  lesions .Of note, this current work had several limitations. Firstly, there could have been a participant selection bias because all the individuals who attended the anal cancer screening consultation were included consecutively. The fact that these patients were attending this consultation was conditioned by the fact that they had a greater interest in undergoing screening for possible anal symptoms suggestive of disease. Secondly, the low number of participants with qualitative HPV-18 could have influenced the interpretation of the results because these data cannot be extrapolated to other contexts and so more patients must be studied in future work.However, despite the limitations of this study, this work is the first of its type to be carried out at a national level in Spain to evaluate and compare the validity of HPV-VL to detect HSILs to that of conventional techniques in anal cancer screening in a population with HIV. While other studies have screened participants with a prior pathological result in anal cytology , with poGiven the wide variability in the results obtained for the predictive capacity of conventional methods such as anal cytology and the qualitative detection of HPV, studying new methods such as HPV-VL quantification can help in the detection of HSILs. This technique is a minimally invasive, well-tolerated test that also eliminates inter-observer variability in the interpretation of anal cytology. Given the overall high sensitivity of viral load quantification reported in this current work, perhaps in the future this technique could be considered a good biomarker for anal cancer screening.versus non-HSILs. All of this reinforces the idea that HPV is implicated in anal cancer and can be used for screening and detection of high-grade lesions. Nonetheless, further studies with more participants will be needed to evaluate the clinical implications that HPV-VL quantification may have in anal cancer screening. Indeed, it would be interesting to conduct longitudinal studies that evaluate HPV-VL at different screening time points in the same patients in order to study the factors that interfere with the progression of the HSILs or the clearance of HPV itself.In conclusion, in our work, quantification of HPV with cut-off values for HPV-16, HPV-18, and HPV-16-18 co-testing of 6.2, 59.8, and 41.7 copies/cell, respectively, improved the sensitivity for the detection of HSILs compared to anal cytology or the qualitative detection of HPV, although it did not improve the specificity. In addition, the HPV-16-VL and HPV-16-18-VL co-testing results were statistically significant for the diagnosis of HSILs 10.7717/peerj.15878/supp-1Supplemental Information 1Click here for additional data file.


Bungarus multicinctus and Daboia russelii venoms via analytical SEC using volatile, non-salt-containing eluents for post-column mass spectrometry, coagulation bioassaying and high-throughput venomics. Two venoms were used to demonstrate the method developed. While the venom of Bungaurs multicinctus is known to exert anticoagulant effects on plasma, in this study, we showed the existence of both procoagulant toxins and anticoagulant toxins. For Daboia russelii venom, the method revealed characteristic procoagulant effects, with a 90 kDa mass toxin detected and matched with the Factor X-activating procoagulant heterotrimeric glycoprotein named RVV-X. The strong procoagulant effects for this toxin show that it was most likely eluted from size exclusion chromatography non-denatured. In conclusion, the separation of snake venom by size gave the opportunity to separate some specific toxin families from each other non-denatured, test these for functional bioactivities, detect the eluting mass on-line via mass spectrometry and identify the eluted toxins using high-throughput venomics.Modern analytical size exclusion chromatography (SEC) is a suitable technique to separate venom toxin families according to their size characteristics. In this study, a method was developed to separate intact venom toxins from  Snakebite envenoming is considered as a neglected tropical disease mainly affecting those living in (sub-)tropical countries in the developing world . Due to 2s (PLA2s), snake C-type lectins (snaclecs), snake venom metalloproteinases (SVMPs), and snake venom serine proteinases (SVSPs) [The two most medically relevant snake families are the Elapidae (elapids) and Viperidae (vipers) . Both of (SVSPs) ,8,9,10,  (SVSPs) .18 column and a gradient with an acidic-organic mobile phase [To determine the individual activities of specific toxins, separation of the crude venom followed by bioactivity measurements is required. In many studies, separation is performed by reversed-phase high-pressure liquid chromatography (RP-HPLC or RPLC) using a common Cle phase . This sele phase ,14,15. Dle phase ,10.2s are between 12 and 15 kDa, separation by size-based chromatography provides a useful separation tool in venom research [Since the total mass of toxins within one family falls in a specific mass range, for example, 3FTxs are between 5 and 8 kDa or PLAresearch . In convresearch ,18. By mresearch . Howeverresearch .Bungarus multicinctus) and the Russell’s viper (Daboia russelii) [The objective of this study was to develop an analytical technique that could be used to separate and fractionate snake venoms by toxin family in their native and/or biologically active state using SEC coupled with MS, followed by bioassaying and high-throughput venomics (HT-venomics ) charactusselii) ,24.D. russelii and B. multicinctus onto 384-well plates, which were then used for HT-venomics and bioassays. SEC-MS was performed post-column in an on-line fashion. The results of the HT-venomics, bioassays, and SEC-MS analysis are displayed and discussed in The first part of this study consisted of the optimisation of the SEC-separation approach, with a core focus on exploring the impact that differences in the mobile phase conditions had on separation . Next, sB. multicinctus and D. russelii. The optimisation steps are elaborated on in the Optimisation of the SEC separations was performed using the protein mix (PM) and venom from As anticipated, different flow rates See  did not B. multicinctus showed similar results when different mobile phases were used. Separations of venom from D. russelii evaluated with different mobile phase compositions were difficult to compare using SEC-UV data. This was due to the many dissimilarities in retention time profiles , with the addition of 0.05–0.1% acidifier, was sufficient for SEC-based separation see . HoweverD. russelii and the elapid B. multicinctus were used to evaluate the application of analytical SEC for venom research. Both species are known to have anti- and/or pro-coagulant venom toxins [The venoms of the viper m toxins ,24 but hHigh-throughput (HT) venomics was performed on SEC-separated and fractionated snake venoms. The identified toxins are presented in chromatograms plotted by the measured protein score against the retention time of fractionation, in so-called protein score chromatograms (PSCs). The data can be found in B. multicinctus venom, a total of 32 Uniprot toxin identities were matched when combining the results for all separately measured 384-well plates containing SEC-fractionated venom. Samples separated using the eluents with the two different TFA concentrations gave the most hits (28 toxins identified), while separations with 0.05% FA in the eluent gave the least hits (22 toxins identified). None of the identified toxins had known coagulation activity according to the sparse functional data associated with the proteins present in the Uniprot database; however, some were described as causing platelet aggregation inhibition which is known to enhance anticoagulation [HT-venomics data obtained for the six different mobile phase compositions were compared. Since the plates with fractionated venom toxins were vacuum-centrifuged to dryness before performing the HT-venomics step, the mobile phases had no influence on the downstream tryptic digestions and subsequent nanoLC-MS/MS measurements. For D. russelii venom, a total of 33 different toxins were detected from all tested SEC separations combined. Most toxins were identified in the fractionated wells from the separation with 0.05% DFA in the eluent (27 venom toxins), and the lowest number of toxins was found for separation with 0.1% TFA in the eluent (19 venom toxins). A toxin from another closely related species, Daboia palaestinae, was also retrieved (with a low protein score) in one of the six fractionated plates (separation by 10% ACN + 0.05% DFA). Toxins in the D. russelii venom predicted to cause both anti- and pro-coagulant venom effects (according to the Uniprot database) were identified from the HT-venomics data. The anticoagulant toxins detected included two PLA2s (PA2B8 and PA2B3) and five snaclecs . Tentative procoagulant toxins found were two snaclecs (SLLC1 and SLLC2), an SVMP (VM3CX), and two SVSPs (VSPA and VSPG).For B. multicinctus venom, aligning the venom SEC-UV separation chromatograms with the bioassay data provided toxin mass ranges where anti- and/or pro-coagulant venom activity occurred, based on the SEC elution time profile. Substantial variations were observed for the coagulation assay results depending on the different separation conditions applied . Because of the large toxin mass range, it was challenging to identify the responsible toxins. However, given that B. multicinctus venom is known to have approximately 4% SVMPs [B. multicinctus venom. However, a coagulation factor X activation toxin is known to be present in the venom of a closely related species, B. fasciatus [Contrastingly, pro-coagulant venom effects were only detected when using the separation conditions containing FA . Although procoagulant venom effects have been detected in several  species ,34, to o4% SVMPs ,36 and mB. multicinctus is caused by SVMP toxins, experiments were repeated in the presence of the metalloprotease inhibitor marimastat. B. multicinctus venom was separated via SEC using 10% ACN + 0.05% FA as an eluent, and marimastat was added to the coagulation bioassay with a final assay concentration of 4 µM. The results of the bioassay in the presence and absence of marimastat are shown in B. multicinctus venom exert procoagulant effects in this bioassay. We also observed a change in anticoagulant venom effects in the presence of marimastat . The identified toxins in 2s or A-chain of a ß-bungarotoxin (code starts with PA) and Kunitz-type serine proteinases or B-chain of a ß-bungarotoxins (VKTH1 and VKTH2). Since there are many different toxins within the retention times for both the coagulation activities, no exact matches could be made, which was expected for the intrinsically low-resolution SEC separations. In the detected procoagulation elution time frame of 9 to 13 min  and HT-venomics data for the SEC separation, using 10% ACN and 0.05% DFA as the mobile phase. In 2s (PA2B3 and PA2B8), snaclecs , SVSPs (VSPA and VSPG), and an SVMP (VM3CX). All plotted PSCs are illustrated separately in 2s (PA2B3 and PA2B8) identified by HT-venomics fall within this range of 12 to 19 min and therefore match with the anticoagulation profile observed for the bioassay performed with marimastat. The potentially anticoagulant snaclecs were also detected, outside of the main anticoagulation range, and were found at different retention time frames within the whole retention time range. Therefore, it is assumed that these are most likely not the main cause of the anticoagulant venom effects observed. The anticoagulation profile in m/z peaks, the total ion currents (TICs), and the extracted ion chromatograms (EICs), can be found in Because we employed watery mobile phases during SEC separation without non-volatile salts as buffer components in the mobile phase , we could connect the SEC effluent directly to MS to obtain intact toxin MS data for the separated toxins in each venom analysed. With a flow splitter integrated in-between the SEC and the MS, we directed 10% of the flow from the SEC output to the MS, enabling us to accurately measure the high-resolution masses of eluting intact toxins. These SEC-MS data were used as validation for the SEC separations of the toxins in the venoms analysed. The toxin masses found were compared to the exact masses of the toxins retrieved by the Mascot searches from the HT-venomics experiments. All MS data, including the deconvoluted B. multicinctus venom resulted in three main peaks and one shoulder peak in front of the first peak in the SEC-UV data for every mobile phase composition. Since ionisation sensitivity by ESI decreases upon mass size, the first eluting largest toxins were only observed in very low abundance. Additionally, it is known from this elapid venom  that larger toxins are only generally present in low abundances [2 subunit (±13.5 kDa) and a B-chain that is a Kunitz-type serine protease inhibitor subunit (±7 kDa) both bound together by cysteine bridges according to Uniprot [2s. Since the peaks were not baseline-separated , it is not unexpected that masses of around 13 kDa are represented in both peaks. The last main peak within the column volume time  only resulted in the detection of around 9 kDa masses when TFA was used in the mobile phase. In this case, secondary interactions with the column are the most likely cause of this. Overall, more toxin masses were detected with a concentration of 0.1% acidifier compared to 0.05% acidifier. The acidifier is necessary for proper ionisation in positive electrospray ionisation mode, which also explains why a higher concentration gives more (intense) peaks. Most toxins were detected with 0.1% DFA, and the least were found for 0.05% FA. Despite FA being the better ioniser according to the literature, in this study, better ionisation performance was clearly obtained in DFA [The separation of undances . When ex Uniprot ,47. The d in DFA .m/z value of 3477.10 with a 26+ charge. The charge state distribution for this peak was determined manually due to low background noise. To our knowledge, the detection of an around 90 kDa peak for D. russelii venom has not yet been shown by ESI-MS but only by MALDI-MS in the past [D. russelii analyses. These masses correspond to the mass range of PLA2s within this venom. The remaining peaks were expected to contain masses <11 kDa, but this was not the case. There were masses of ±13 and ±27 kDa that were eluted here and even after the column inclusion end time of 21 min. These compounds most likely interacted with the column material and were thus not separated solely based on their size. From the SEC-UV data, a peak was also observed at 28 min retention time (RT), but for this peak, no masses were found in the MS data.For each acidifier, slight variations in resulting peak distribution were observed. This, for example, resulted in non-baseline separated peaks in the case of separation by 10% ACN + 0.1% FA and 10% ACN + 0.05% FA where two peaks from the SEC-UV chromatogram were fused into one broad TIC peak. Despite these differences, a comparison could be made between eluting masses for all separations tested. No significant masses in the venom toxin range could be derived from the first peak in the TIC for separations with TFA and DFA. The second peak in the TIC for these separations gave 2–5 toxins with masses of around 25 or 29 kDa. The first two peaks of the separations with TFA or DFA were fused into one broad peak in the TIC of separations by FA. From this fused peak for separations by FA, masses of ±25, ±29, ±31, and ±90 kDa were extracted. The highest detected accurate mass was 90,378.34 Da, calculated from an the past . This mathe past . The nexB. multicinctus than for D. russelii venom, likely because toxin types abundant in B. multicinctus venom (such as most 3FTx and PLA2s) lack PTMs.The HT-venomics data were next matched with the parallel obtained SEC-MS data. Matches were made based on the calculated mass, peak shape, and retention time of the PSCs and the EICs. For toxins without post-translational modifications (PTMs), masses derived from the SEC-MS data could directly be matched with the masses of the toxins calculated from their sequence. A PTM such as glycosylation alters the mass, and, therefore, matching the exact deconvoluted masses from SEC-MS to data retrieved from the HT-venomics experiments is challenging. However, it was possible to match several masses to specific toxins see , with moB. multicinctus venom, which is consistent with the mass range of ß-bungarotoxins, which are composed of a PLA2 chain (A-chain) and a Kunitz-type serine proteinase inhibitor chain (B-chain), covalently linked via a cysteine bridge. These two chains were individually identified in the HT-venomics data, where nine A-chains and three B-chains were identified. According to Uniprot, any A-chain can be bound to any other B-chain. Therefore, the exact masses from the SEC-MS data were not matched with one 20–21 kDa toxin from the HT-venomics data but could only be matched with an A-chain of ±15 kDa and a B-chain of ±7 kDa combined. After making calculations, two detected masses from the SEC-MS data could be matched with an A- and B-chain combined, with a difference in mass of ±3 Da (see 2 ß-bungarotoxin A2 chain (Uniprot code PA2B2), of which, the exact mass derived from the Uniprot database matched one mass calculated from the SEC-MS EIC , showed an unusual peak shape, appearing as a first small followed by a second larger peak, in both the EIC from the MS data and in the corresponding PSC see . The firD. russelii venom, toxins were found within the mass range of 11 to 14 kDa. This venom contains several PLA2s of approximately 13 kDa. No toxins were detected by HT-venomics with around 11 to 12 kDa masses  that has a covalently bound heterotrimeric structure [D. russelii using 10% ACN + 0.05% FA as the mobile phase  matched with the bioassay procoagulation peak. The small shoulder peak in the EIC at time 11.5–15 min explained the extended procoagulation observed. Earlier, in tructure  and a prtructure ,51. Howetructure . The indhase see . From FiIn the same figure, B. multicinctus venom and achieved on-line MS detection of a 90 kDa mass D. russelii heterotrimeric toxin with SEC-MS. Contrastingly, TFA and DFA showed better baseline-separated peaks in the resulting SEC-UV data. The ESI-MS results showed that some toxins, predominantly PLA2s in D. russelii venom, eluted later than most of the other toxins within the same mass range. Therefore, it is important to take into consideration that the SEC column is not always suitable for a perfect size-based separation of all toxins in any given venom. Whether this is a phenomenon that occurs specifically with the SEC column used in this research needs to be tested further by comparison of different analytical SEC columns.The aim of this study was to develop an analytical method to separate snake venom toxins based on their size while retaining their biological activity, using post-column on-line MS analysis. An analytical SEC method using HPLC with low concentrations of organic acidic mobile phases was optimised to perform the separation. A post-column HTS coagulation bioassay provided information about the coagulation-modulating toxins in the snake venoms, where parallel HT-venomics was used to identify these specific toxins. ESI-MS gave accurate masses of eluting toxins and provided information about the capacity of the separation via SEC based on toxin mass. Toxins such as SVMPs and SVSPs tend to lose their bioactivity when separated by the more often used reversed-phase HPLC because of possible alterations in their native structure caused by either column interactions or the high concentrations of organic solvents used in the mobile phase. From the organic solvent modifiers tested, IPA gave high column back-pressures due to its high viscosity and caused peak splitting. ACN (10%) with the addition of an acidifier was found to be the best organic modifier and concentration for the mobile phase. The separations differed slightly from each other, with three acidifiers tested at concentrations of both 0.1% and 0.05%. FA gave the most promising results when analysing the downstream bioassays and MS data, as it was the only condition that enabled the detection of procoagulant activity caused by B. multicinctus venom has not been described before, and the inhibition of this activity by marimastat suggests that it is mediated by SVMP toxins. Toxins in D. russelii venom consistently showed both anti- and pro-coagulant activity. HT-venomics was used to gain information on the identity of these coagulopathic toxins, confirming the presence of the heterotrimeric procoagulant toxin RVV-X and demonstrating that it remained intact following SEC separation. Collectively, our data demonstrate that it is feasible to separate snake venom by size using an analytical SEC approach coupled on-line with ESI-MS. Venom HT-venomics and ESI-MS can be performed in parallel and in combination with post-column bioassaying to enable simultaneous functional characterisation and identification of venom toxins that are susceptible to denaturation via more commonly employed reverse-phase separation approaches.To our knowledge, procoagulant venom activity by For the SEC optimisation and calibration steps, a protein mix (PM) was made from proteins 1 mg/mL bovine serum albumin (BSA) (mass of 66.5 kDa) and 1 mg/mL ribonuclease A (RNase A) (mass of 13.7 kDa), and nucleotide 0.1 mg/mL uracil (mass of ±100 Da), all purchased from Sigma-Aldrich, dissolved in Milli-Q water, kept cold at 4 °C, and used within one month. Uracil was added as the inclusion limit measurement. The organic solvents used for the optimisation of the mobile phase were acetonitrile (ACN) (Biosolve) and isopropanol (IPA) (Sigma-Aldrich) at eluent concentrations of 5–20% diluted in Milli-Q. Trifluoroacetic acid (TFA), formic acid (FA) (Biosolve), and difluoroacetic acid (DFA) (Sigma-Aldrich) were added to the eluents at final concentrations of 0.05% or 0.1%. All reagents were of analytical grade. For the coagulation assay, 20 mM calcium chloride was prepared by dissolving calcium chloride (Sigma-Aldrich) in Milli-Q water. Bovine plasma used for this assay was purchased from Biowest. The metalloproteinase inhibitor marimastat was purchased from Sigma-Aldrich.Bungarus multicinctus and Daboia russelii were sourced from the historical collection at the division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam (VU). The venoms were stored at −80 °C in their lyophilised form, brought into solution with Milli-Q in Eppendorf tubes at concentrations of 1, 2.5, and 5 mg/mL, aliquoted to the required volumes, and stored again at −80 °C until further use. At the time of analysis, the venoms were taken from the freezer, kept at 4 °C, and used within one week.Venoms from m/z. Other parameters were 500 V for the end plate offset, 4500 V for the capillary, 1.5 bar for the nebuliser, 8 L/min dry gas, and 200 °C as the dry temperature. MS calibration was performed using an ESI-L low-concentration tune mix (Agilent). From the on-line SEC-MS data, the total ion chromatograms (TICs) and extracted ion chromatograms (EICs) for each venom separated with every different mobile phase solution used were plotted and depicted in two PowerPoint documents. The TICs and EICs for B. multicinctus are visualised in ‘PP S1 SEC-MS B. multicinctus’, and for D. russelii in ‘PP S2 SEC-MS D. russelii’.Size exclusion chromatography was performed using a 4.6 × 300 mm Sepax Zenix SEC-300 column with a 3 µm particle size and 300 Å pore size. The column was operated at 30 °C during the separations using a Shimadzu CTD-30A column oven. The flowrate and injection volumes were varied at first to observe their effects, whereafter the most fit parameters were chosen. Separation was performed using a Shimadzu LC20AB system with a dual pump. The high-performance liquid chromatography (HPLC) system was connected to a Shimadzu SPD 20A UV/Vis detector. Detection was performed at wavelengths of 220, 254, and 280 nm, respectively, where 280 nm was used for HPLC-UV data analysis. HPLC-UV data was acquired by and processed with Shimadzu LabSolutions software and was exported to PRISM software for plotting the HPLC-UV chromatograms. For electrospray ionization mass spectrometry (ESI-MS) analysis, a Bruker Maxis II quadrupole time-of-flight (Q-TOF) mass spectrometer was used. SEC-MS was performed online where a fraction of 10% from the HPLC effluent was sent to MS using a post-column flow splitter. An electrospray ionisation (ESI) source was applied in positive mode and ions were scanned in a mass range of 800–5000 TM FRM100 nanofraction collector (SPARK-Holland and Vrije Universiteit Amsterdam). A fractionation resolution of 12.5 s was used, resulting in approximately 37.5 µL eluent collected per well at a flow rate of 0.2 mL/min. Within 1 h after fractionation, plates containing volatile eluent were vacuum-centrifuged overnight using a Christ Rotational Vacuum Concentrator RVC 2-33 CD plus  and kept at −20 °C until further use. The delay time between the UV/Vis-detector and the fractionation system was measured by injecting 10 µL of 0.1 mg/mL argatroban, fractionating this anticoagulant compound after separation, and then performing the plasma coagulation assay on the plate containing the fractionated argatroban. The resulting negative peak in the bioassay chromatogram was subsequently compared to the argatroban peak observed in the UV/Vis chromatogram from which a delay of 0.6 min was observed.High-resolution fractionation was performed during the HPLC runs, after the flow split, using 90% of the effluent portion for fractionation into transparent flat-bottom polystyrene 384-well plates , making use of a FractioMateTo identify the SEC-separated toxins, venom proteomics  was performed using the methodology of Slagboom et al., 2023 . This wam/z. MS/MS was performed via collision-induced dissociation (CID) in data-dependent mode using 10 eV collision energy. Data were collected and processed by Bruker DataAnalysis software. Finally, the identification of the toxins was performed using MASCOT (Matrix Science) searches against Swiss-Prot and species-specific databases using the same search conditions as Slagboom et al. in 2023 [B. multicinctus and PRISM S9-S14 for D. russelii.The analytical part of the HT-venomics procedure was accomplished by nanoLC-MS/MS. The digested samples were separated by an UltiMate 3000 RSLCnano system . This system used an autosampler which allowed direct sampling from the 384-well plates. A volume of 1 µL was injected in a C18 nanoLC column (150 mm × 75 µm) with a 2 µm particle size and 100 Å pore size, coupled with a trapping C18 column (5 mm × 0.3 mm) with a 5 µm particle size and 100 Å pore size. Both columns were from Acclaim™ PepMap™ 100 (Thermo Fischer Scientific). The column oven was set to 45 °C. The mobile phase included eluent A (2% ACN and 0.1% FA in Milli-Q) and eluent B (98% ACN and 0.1% FA in Milli-Q), both used in a gradient program. This gradient was: 3 min isocratic separation at 1% B, linear increase to 40% B in 7.5 min, linear increase to 85% B in 0.1 min, isocratic separation at 85% for 0.7 min, linear decrease to 1% B in 0.2 min and isocratic separation at 1% B for 3.7 min. A flow rate of 5 µL/min was used. The nanoLC was connected to a MaXis II Q-TOF MS operated in positive-ion mode. The nanoESI source had the following parameters: capillary voltage, 1.6 kV; source temperature, 150 °C; gas flow, 3.0 L/min; and nanoBooster pressure, 0.20 bar. The mass range was set from 50 to 3000  in 2023 . The pro2+ results in a kinetic coagulation profile measured by spectrophotometric instrumentation, as upon coagulation, the plasma becomes less transparent, hence resulting in higher absorbance. The coagulation bioactivity of crude venoms and venom toxins can be measured by testing the influence on the coagulation profile kinetically . Whether a compound is activating or inhibiting the coagulation process depends on the velocity of the plasma clotting. A high velocity, meaning a fast increase in absorbance and steep slope, is caused by a procoagulant toxin, while an anticoagulant toxin is responsible for a slow increase in absorbance.An HTS (high-throughput screening) coagulation assay described by Still et al., 2017 was utilised in this study to measure the effect on plasma coagulation caused by different toxins in the fractionated samples of the analysed venoms ,37. BoviB. multicinctus and PRISM S9-S14 for D. russelii.To reduce the potential for different toxins coeluting after separation simultaneously causing competing bioactivities , we also used the small-molecule-inhibiting drug marimastat  to investigate the effect of inhibiting procoagulant SVMPs after chromatographic separation. The concentrations of marimastat used were selected based on Xie et al., 2020 . Inhibit


Mn-CH7D gene was highly expressed in the ovary, which was much higher than that in other tissues, and the expression level of Mn-CH7D reached the highest level at the third stage of the ovarian development stage (O-III). During embryonic development, the Mn-CH7D gene expression was highest in the zoea stage. The function of the Mn-CH7D gene was explored by RNA interference. The experimental group was injected with Mn-CH7D dsRNA through the pericardial cavity of M. nipponense, while the control group was injected with the same volume of dsGFP. Statistical analysis of gonadal development and GSI calculation showed that the silencing of Mn-CH7D resulted in the suppression of gonadal development. In addition, the molting frequency of the experimental group was significantly lower than that of the control group during the second molting cycle after silencing Mn-CH7D. On the seventh day after silencing, ecdysone content in the experimental group was significantly reduced. These results demonstrated that the Mn-CH7D gene played a dual role in ovarian maturation and molting of M. nipponense.The Cholesterol 7-desaturase gene plays an important role in insect ecdysone synthesis, but its role in ovarian development has not been reported. In this study, characteristics and the phylogenetic relationship of Cholesterol 7-desaturase were identified by bioinformatics. qPCR showed that the  Macrobrachium nipponense  is an economically important freshwater aquaculture species in China and the only indigenous species in large-scale aquaculture freshwater shrimp  domain and non-heme iron binding domain. The Rieske [2Fe-2S] domain and non-heme iron binding domain are involved in electron transfer and are essential for catalytic enzyme function [Mn-CH7D itself has an enzymatic activity on sterols. Mn-CH7D can convert cholesterol into 7-dehydrogenated cholesterol, and many isoenzymes exist in insects and crustaceans, such as DAF-36 [NVD [Mn-CH7D gene clustered into one branch in crustaceans and insects, indicating that Mn-CH7D is more conserved in the same class.Ecdysteroids regulate many aspects of the developmental process and reproductive activity in insects and crustaceans ,26,27. Cfunction . It is, s DAF-36  and NVD -36 [NVD ,33,34. TM. nipponense actively synthesized ecdysteroids, consistent with the previous reports [Mn-CH7D peaked at O-III, which verified that Mn-CH7D was closely related to ovarian maturation [M. nipponense, suggesting that it is likely involved in the regulation of molting [Mn-CH7D increased sharply from the nauplius stage (NS) to the zoea stage (ZS) and peaked at ZS. At the zoea stage, the embryonic development of M. nipponense was basically completed [Mn-CH7D played a critical role in hatching. Similar results were reported in the study of Penaeoidean shrimp Sicyonia ingentis by Chang E S et al., which found that in Penaeoidean shrimp, the content of molting steroid is very little after laying eggs, and it begins to increase in the embryonic development process. And it reached the maximum in the pre-hatching stage [Mn-CH7D began to increase until L10, and the expression level of L10 was the highest in the whole process of larva development, indicating that the function of Mn-CH7D gene is closely related to ontogeny, especially larva development. After metamorphosis, the expression level of Mn-CH7D reached the highest on the 15th day after metamorphosis (PL15), and there was no significant difference in other stages. At PL15, the gonads began to differentiate, suggesting that Mn-CH7D was associated with gonad development.Tissue-specific analysis revealed that transcripts were detected at much higher levels in the ovaries of female adults than in other tissues, suggesting that the ovary of  reports . The expturation . Previou molting . qPCR atompleted , indicatng stage . After mMn-CH7D in reproductive processes, we knocked down Mn-CH7D using RNAi. The expression of Mn-CH7D in the ovaries of M. nipponense was significantly reduced by injection of dsRNA on day 4  . The level of ecdysone tends to increase on day 7, as the second molt begins on day 14, and the level of ecdysone will increase before molting, which has been demonstrated by previous studies [Mn-CH7D gene inhibited the synthesis of ecdysone, thereby affecting the molting of M. nipponense. Cholesterol is catalyzed to the active ecdysone by a series of P450 enzymes. Similar results were observed that the knockout of Spook and CYP302a1 could inhibit molting and reduce the content of ecdysone [Bemisia tabaci, Bombyx mori, and Locusta migratoria [After RNAi, the molting frequency of the experimental group was significantly lower than that of the control group on days 16–20 ( < 0.05) . Thus, s by RNAi . After t studies ,43. Thesecdysone ,45. It hgratoria ,47,48.Mn-CH7D gene is an ecdysone synthesis pathway gene, which is involved in ecdysone synthesis and plays an important role in the molting and ovarian maturation of crustaceans. This study demonstrated for the first time that Cholesterol 7-desaturase involved in ecdysone synthesis act as gonadotropin in M. nipponense, providing a theoretical basis for the artificial control of ecdysone. At the same time, it also provides strong evidence that this ecdysone is a key hormone in controlling reproductive ecdysone. These results strongly suggest that the M. nipponense (body weight 0.56 ± 0.13 g and body length 3.68 ± 0.22 cm) were obtained from Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu Province, China. No endangered or protected species were involved in this experiment. All experimental protocols and methods were approved in September 2022 (Authorization no. 20220901002) by the Animal Care and Use Ethics Committee in the Freshwater Fisheries Research Center . The prawns were raised in an indoor circulating aquaculture system  to acclimate for one week. During the culture period, the water temperature was maintained at about 27 ± 1 °C. M. nipponense were fed a daily ration of paludina in the morning and evening. Each feeding amount depended on the weather, feeding conditions, timely adjustment, and clean food scraps once a day ensured water quality.Healthy female M. nipponense were gathered, frozen immediately in liquid nitrogen, and then stored at −80 °C until use. Individuals of M. nipponense at different developmental stages  were also dissected, frozen, and stored at −80 °C separately for future experiments. The specific classification of ovarian stages and different stages of the embryo is listed in Tissues of the eyestalk, brain, heart, hepatopancreas, gills, muscles, and ovaries of female M. nipponense at different developmental stages and different tissues was extracted using RNAiso Plus reagents  according to the manufacturer’s instructions, and quality was assessed by 1% agarose gel electrophoresis. The first-strand cDNA was synthesized with RNA as a template using the M-MLV reverse transcriptase  kit at 42 °C for 2 min. The reaction was finished after 37 °C for 5 min and 85 °C for 30 s. Then, the synthesized cDNA was kept at −20 °C for subsequent quantitative real-time PCR (qPCR) reaction to detect the expression pattern of the Mn-CH7D gene in M. nipponense. EIF was used as the internal reference gene [Mn-CH7D mRNA was calculated by the 2−△△CT method [The total RNA of nce gene . The conT method .Mn-CH7D cDNA sequence determined by combining a subset of sequences from the M. nipponense hepatopancreas transcriptome library with the 3′race production was obtained. According to the Cholesterol 7-desaturase sequence, the primers used were designed by a prime designing tool  (The full-length DNA sequence of Cholesterol 7-desaturase was cloned using a 3′RACE kit according to the manufacturer’s instructions . The fulay 2022) .https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 5 May 2022); amino acid sequence alignment was analyzed by DNAMAN 6.0 software; the open reading frame (ORF) of CH7D was predicted by the ORF Finder program ; signal peptide was predicted using Signal 5.0 Server . Molecular weight (MW), the isoelectric point (PI), and amino acid composition were performed using ProtParam on the ExPASy website . The phylogenetic tree was constructed based on the Cholesterol 7-desaturase protein and its orthologs in other species by the neighbor-joining (NJ) method using the MEGA 7.0 software.Domains in the protein sequence were determined via Conserved Domain Searches at NCBI  with DIG signal were designed by Primer5 software and synthesized by Shanghai Sangon Biotech Company. More details of in situ hybridization have been described in previous studies [Ovarian (stage I–V) samples fixed in 4% paraformaldehyde solution were used for in situ hybridization study. Based on the cDNA sequence of  studies . The prohttps://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl, accessed on 1 May 2022). The dsRNA of Mn-CH7D was synthesized using Transcript Aid™ T7 High Yield Transcription kit , and its concentration was measured/determined at 260 nm using a BioPhotometer . Expression levels of Mn-CH7D were measured by qPCR to determine the interference efficiency. The primers used to synthesize double-stranded RNA (dsRNA) were designed with the online software Snap Dragon  through the pericardial cavity of M. nipponense [dsGFP. The frequency of injections was five days. The Mn-CH7D expression of the ovary was investigated to detect the interference efficiency by qPCR.A total of 300 healthy female prawns in stage IV were randomly distributed into 6 aquaculture tanks in equal proportion, which were the experimental group and the control group. The experimental group was injected with pponense , while tThe ovarian development was observed and recorded daily. The specific classification criteria are shown in The number of molts was observed and recorded daily. Molting frequency is calculated using the following formula: Molting frequency = (Nm/Ns)/D, where Nm is the total number of molts, Ns is the number of prawns in the aquarium, and D is the number of experimental days.Three prawns were randomly selected from experimental and control groups on the 1st, 7th, 14th, and 20th day after injection, respectively. These shrimp were tested for hormone contents. The contents of ecdysone in prawns were determined by enzyme-linked immunosorbent assay with the double antibody two-step sandwich method, according to the Shrimp EHELISA Kit instruction. Experiments were performed in triplicate.At the end of the experiment, the ovaries of two shrimp from the experimental group and the control group were collected and fixed with 4% paraformaldehyde solution for tissue section preparation. The tissue slides were deparaffinized and hydrated using standard procedures and stained with hematoxylin and eosin (H&E). The final observation is a cross-section of the tissue.−ΔΔCt comparative CT method.All quantitative data were presented as mean ± standard deviation (Mean ± SD). SPSS Statistics 24.0 was used for data analysis of the experimental data. The significant differences between the control group and the treatment group were analyzed by one-way ANOVA and Duncan test for multiple comparisons. The significance level of data variance was set at 0.05. The relative mRNA expression levels of the genes were calculated according to the 2M. nipponense was successfully cloned and analyzed. The expression characteristics of Cholesterol 7-desaturase in different tissues and different stages of ovary and embryo development of M. nipponense were investigated. Importantly, this study demonstrated for the first time that this gene involved in ecdysone synthesis act as gonadotropin in M. nipponense, providing a theoretical basis for the artificial control of ecdysone. It also provides some new insights and ideas to solve the production problem of rapid sexual maturation, which is of great significance to the development of the crustacean industry.In this study, the full-length cDNA sequence of Cholesterol 7-desaturase from


The quality of recovery-15 (QoR-15) is a patient reported outcome questionnaire that measures the quality of recovery after surgery and anesthesia. The QoR-15 has been validated in many languages; Turkish version of the QoR-15 has not yet been established. The aims of this study were to translate the QoR-15 questionnaire into Turkish and to perform a full psychometric evaluation of the Turkish version.After translating the original English version of the QoR-15 scale into Turkish, the QoR-15T scale was psychometrically validated. This process included validity, reliability, responsiveness, feasibility. The QoR-15T was evaluated before the surgery and 24 h after surgery.A total of 210 patients completed the pre- and postoperative questionnaires, providing a completion rate of 93.75%. The correlation coefficient between QoR-15T score and VAS score was 0.644 on postoperative day 1 (p < 0.001). Inter item Cronbach’s alpha was 0.863. Global test-retest concordance coefficient was 0.98 (95% CI: 0.94–1.00).The QoR-15T scale is a reliable and valid instrument for evaluating postoperative quality of recovery in Turkish speaking patients. The psychometric characteristics used to assess postoperative quality of recovery were similar to those in the English version. Recovery is a multidimensional and complex process influenced by a variety of factors such as patient characteristics, surgical procedures, and anesthesia . The greMyles et al. developed the quality of recovery - 40 score (QoR - 40) in 2000, and it is now widely used . In 2013The aim of this study was to develop the Turkish version of QoR-15 (QoR-15T) by a translation and cultural adaptation process and to evaluate the validity, reliability, and responsiveness of the QoR-15T for Turkish patients who receive general anesthesia. The authors hypothesized that the QoR-15T would have as comprehensive validity, reliability, and responsiveness as the original English version.ClinicalTrials.gov . The study was conducted in accordance with the principles of the Declaration of Helsinki  vs. < 70 mm [poor]) .Reliability was measured for consistency of the QoR-15T. Reliability was assessed by internal consistency , split-half reliability, and test-retest reliability. The latter was tested between two repeated measurements in a convenience sample of 20 patients 60 min after the postoperative QoR-15T test. The floor and ceiling effect were determined by calculating if 15% of respondents had either the highest (150) or lowest possible score (0).Standardized response mean (SRM) and Cohen’s effect size were used to assess responsiveness. The SRM was calculated as the change scores divided by the SD of the change scores. The Cohen effect size was calculated as the average change scores (from preoperative to postoperative) divided by the SD at baseline. The patient recruitment rate and successful completion rate were used to assess the clinical feasibility of the QoR-15T.There is no standard approach to determine the sample size in scale validity and reliability studies. It is typically recommended that the minimum number of participants is 150 or that the scale be administered to 10–20 times the number of items on the scale ,20. To aThe measurement data were presented as mean ± SD values and categorical data as number (n) and percentage (%). The normal distribution of the continuous variables was tested using the Kolmogorov–Smirnov test. Associations were measured using Spearman rank (ρ) correlation coefficient. Internal consistency was measured using Cronbach α. Test-retest reliability was measured using the intraclass correlation coefficient. Changes from baseline were compared using the signed ranks test. The student’s t-test was used to compare data with normal distribution, and the Mann–Whitney U-test was applied to data not showing normal distribution, and the Chi-square test was used to compare categorical data. All statistical analyses were performed using SPSS version 25.0 for Windows software . The null-hypothesis was rejected if two-tailed p < 0.05.Of the 234 eligible patients, 10 refused to participate in this study, resulting in a 95.7% (224 / 234) recruitment rate. Fourteen patients were discharged within 24 h after surgery; thus, the completion rate at 24 h after surgery was 93.8% (210 / 224) . The meaTo assess for convergent validity, the correlation was evaluated between the QoR-15T and the VAS for recovery. The Spearman rho (ρ) correlation coefficient was 0.644 on the postoperative first day following surgery (p < 0.001). A strong correlation was determined between QoR-15T and VAS (correlation > 0.60). The correlation of each domain of QoR-15T with VAS was calculated. The VAS score was determined to be weakly correlated with psychological support (correlation < 0.30) and moderately correlated with other domains (correlation 0.30–0.60) (p < 0.001). The correlations of postoperative VAS and each domain of the QoR-15T are shown in A negative, moderate (ρ = −0.410), statistically significant correlation was determined between QoR-15T and surgical time (p < 0.001). A negative, moderate (ρ = −0.380), statistically significant correlation was determined between QoR-15T and PACU length of stay (p < 0.001). The QoR-15T and subscale scores were compared according to sex, ASA physical status, education level, and extent of surgery. A statistically significant difference between the scores of males and females was only seen in the subscale of psychological support . In terms of ASA physical status, a statistically significant difference was seen only in the subscale of psychological independence (p = 0.04). No statistically significant differences were seen between the variables of education levels. There was a statistically significant difference between minor-intermediate surgery and major-complex surgery in respect of the global QoR-15T scores . In the variable of grade of surgery, a statistically significant difference was observed between the minor-intermediate and major-complex scores in the psychological support subscale  and in the psychological independence subscale .Patients with a good or poor postoperative recovery, as indicated by a VAS value of ≥ 70 or < 70 mm, were compared to establish discriminant validity. The QoR-15T score was significantly different between these groups  and , (p < 0.0001).Reliability was evaluated by Cronbach α for internal consistency. Inter item Cronbach α was 0.863, and interdimension Cronbach α was 0.755. The inter item and inter dimension correlation matrices for QoR-15T at 24 h postoperatively are shown in Changes in the preoperative and postoperative QoR-15T and responsiveness are presented in This study was conducted to assess the psychometric properties of the QoR-15T and to compare these with the VAS values in adult patients undergoing elective surgery under general anesthesia. The study results revealed that QoR-15T is a valid, reliable, responsive, and easy to use instrument for the assessment of postoperative recovery in the Turkish population.To assess convergent validity, the QoR-15T for recovery was compared with the VAS values. Convergent validity showed that the correlation coefficient between the QoR-15T score and VAS exceeded the published recommendation ρ > 0.60), which was similar to the coefficient in the original study [0, which There was an inverse correlation between QoR-15T and surgery time and PACU time. These correlations were weaker than those mentioned in the original study . In otheThe discriminate validity of the QoR-15T was determined by comparing the scores of patients who had minor and intermediate surgery to those who had major and complex surgery, with the latter scoring significantly lower. Patients with a good or poor postoperative recovery, as determined by the VAS score, were compared to demonstrate discriminant validity. The VAS score was used to classify good and poor recovery, as it is a more objective assessment than the clinician’s or patient’s opinion ,9,10.Cronbach alpha and split-half reliability were used to analyze internal consistency. These two coefficients were both > 0.7, which satisfied the published recommendations. The QoR-40T scale inter item correlation coefficients ranged from 0.40 to 0.76, with item 7 having the lowest value. Although the original scale included groups of negatively correlated binary items, the QoR-15T scale did not have negative correlated binary items. The interdimensional correlation coefficients ranged from 0.59 to 0.86, with psychological support scoring the lowest and physical comfort scoring the highest. There was no negative correlation between binary subdimensions, or between binary items. These data were sufficient to conclude that the QoR-15T has adequate reliability.The Cronbach alpha value of the QoR-40T scale (0.936) is higher than the Cronbach alpha value of the QoR-15T scale (0.863). Both are higher than 0.7, indicating that QoR-15T can be used in place of QoR-40T to achieve the same reliability in a shorter time. As a result of the reliability analysis of the QoR-15T scale, as the corrected item-total correlations of all the items were greater than 0.3 (range: 0.355 to 0.714), it can be concluded that all the items adequately contribute to reliability. The results of test-retest reliability of the QoR-15T indicated that QoR-15T has an acceptable level of reliability. For the test-retest, the value of Cronbach α was 0.98, which exceeds the established criterion of 0.7 for good reliability.Cohen effect size and SRM values > 0.5 suggest a moderate intervention impact, and values > 0.8 suggest a strong intervention impact ,22. In tThe acceptability and feasibility of the QoR-15T was assessed using recruitment rate, completion rate, and time taken to complete the questionnaire. There was a high rate of participation and successful completion, and most patients were able to complete the questionnaire in less than 4 min. Since the completion times for the QoR-40T scale are not specified, it cannot be said what improvement the QoR-15T scale brings in terms of time. Less than 4 min has been shown to be a reasonable time to measure the quality of the review in clinical studies.During the implementation of the QoR-15T scale, certain difficulties were observed. When moving from question 10 to question 11, the inversion of the Likert scale caused confusion.It was also observed that, as the patient transitioned from part A to part B, the practitioner had to assist the patient by stating 0 for “worst possible scenario” and 10 for “best possible scenario”. As previously stated, this was the most negative aspect of the QoR-15T scale. In addition, the fact that there are two questions (items 11 and 12) on the scale to assess pain can create difficulty in understanding. In the current study, patients referred to “tolerable pain” for moderate pain and “terrible pain” for severe agonizing pain.There were several limitations in this study, since it was conducted in a single, tertiary level hospital in Turkey. The first limitation was that, as the study included patients undergoing elective surgeries under general anesthesia, caution was required when applying the QoR-15T to emergency surgery. Second, patients undergoing outpatient surgery, cardiac, and otolaryngological surgery were not included. Finally, patients with poor understanding of Turkish and serious preexisting medical conditions were excluded.In conclusion, the QoR-15 was translated into Turkish, and its usefulness was assessed in surgical patients who underwent various surgeries under general anesthesia. The results demonstrated that the QoR-15T is a valid, reliable, feasible, and responsive method of assessing postoperative recovery. When compared to the QoR-40T, the QoR-15T provides an equally comprehensive but less time-consuming assessment of a patient’s QoR following anesthesia and surgery. These results support the use of the QoR-15T during the perioperative period to evaluate patient recovery after anesthesia and surgery.